This project will investigate the anatomical circuitry involved in the control of corticotropin releasing factor (CRF) secretion into the hypophysial portal system. It is well established that the release of CRF into the portal blood supplying the pituitary causes the release of adrenocorticotropin and beta-endorphin. These, in turn, begin a cascade of endocrine responses to stresses being experienced by the organism. Together, these represent one of the most important adaptive mechanisms for maintaining a relatively constant internal milieu in the face of various internal or environmental stresses. Recently, CRF neurons projecting to the neurohemal zone of the median eminence have been localized within the various subnuclei of the hypothalamic paraventricular nucleus (PVN). The PVN is involved in intergrating autonomic and endocrine responses that maintain homeostasis, and detailed light microscopic studies have demonstrated that the circuitry within the nucleus is highly differentiated. While light microscopic studies may suggest the PVN is highly differentiated and that certain neuronal interactions may exist with CRF neurons, the full degree of differentiation and actual synaptic relationship require detailed immunocytochemical studies at the ultrastructural level. Using a variety of techniques, including peptide immunocytochemistry, fluorescent retrograde and lectin anterograde neuronal tract tracing, and a new double-labeling method for electron microscopic immunocytochemistry, we will determine the anatomical interactions of axons from five immunocytochemically defined neuron types with CRF neurons of the paraventricular nucleus. The five neuron types (dopamine, norepinephrine, epinephrine, gamma-aminobutyric acid, adrenocorticotropin) have been shown previously, pharmacologically and/or anatomically, to potentially influence CRF neurons of the PVN. We will: 1) determine the distribution of each fiber type within the various subnuclei of the PVN, especially their relationship to CRF neurons; 2) determine the cells of origin for these axons; and 3) determine the synaptic interaction of these fiber types with CRF neurons of the PVN. This later goal will use a powerful new method for the simultaneous labeling of multiple antigens at the ultrastructural level. The method, developed in our laboratory, calls for the labeling of anti-CRF with moderate numbers of small atomic weight elements and then localizing the antigen-antibody reactive site in an analytical electron microscope using electron spectroscopic imaging.